Role Of Cell Cycle Proteins Research Articles

Selective Autophagy Regulates Cell Cycle in Cancer Therapy.

Aberrant function of cell cycle regulators results in uncontrolled cell proliferation, making them attractive therapeutic targets in cancer treatment. Indeed, survival of many cancers exclusively relies on these proteins, and several specific inhibitors are in clinical use. Although the ubiquitin-proteasome system is responsible for the periodic quality control of cell cycle proteins during cell cycle progression, increasing evidence clearly demonstrates the intimate interaction between cell cycle regulation and selective autophagy, important homeostasis maintenance machinery. However, these studies have often led to divergent rather than unifying explanations due to complexity of the autophagy signaling network, the inconsistent functions between general autophagy and selective autophagy, and the different characteristics of autophagic substrates. In this review, we highlight current data illustrating the contradictory and important role of cell cycle proteins in regulating autophagy. We also focus on how selective autophagy acts as a central mechanism to maintain orderly DNA repair and genome integrity by degrading specific cell cycle proteins, regulating cell division, and promoting DNA damage repair. We further discuss the ways in which selective autophagy may impact the cell cycle regulators, since failure to appropriately remove these can interfere with cell death-related processes, including senescence and autophagy-related cell death. Imbalanced cell proliferation is typically utilized by cancer cells to acquire resistance. Finally, we discuss the possibility of a potent anticancer therapeutic strategy that targets selective autophagy or autophagy and cell cycle together.

Cell cycle proteins as promising targets in cancer therapy.

Cancer is characterized by uncontrolled tumour cell proliferation resulting from aberrant activity of various cell cycle proteins. Therefore, cell cycle regulators are considered attractive targets in cancer therapy. Intriguingly, animal models demonstrate that some of these proteins are not essential for proliferation of non-transformed cells and development of most tissues. By contrast, many cancers are uniquely dependent on these proteins and hence are selectively sensitive to their inhibition. After decades of research on the physiological functions of cell cycle proteins and their relevance for cancer, this knowledge recently translated into the first approved cancer therapeutic targeting of a direct regulator of the cell cycle. In this Review, we focus on proteins that directly regulate cell cycle progression (such as cyclin-dependent kinases (CDKs)), as well as checkpoint kinases, Aurora kinases and Polo-like kinases (PLKs). We discuss the role of cell cycle proteins in cancer, the rationale for targeting them in cancer treatment and results of clinical trials, as well as the future therapeutic potential of various cell cycle inhibitors.

Physiological and pathophysiological functions of cell cycle proteins in post-mitotic neurons: implications for Alzheimer's disease.

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disorder for which no effective treatment is available. Increased insight into the disease mechanism in early stages of pathology is required for the development of a successful therapy. Over the years, numerous studies have shown that cell cycle proteins are expressed in neurons of AD patients. Traditionally, neurons are considered to be post-mitotic, which means that they permanently retract from the cell cycle. The expression of cell cycle proteins in adult neurons of AD patients has therefore been suggested to promote or even instigate pathomechanisms underlying AD. Interestingly, expression of cell cycle proteins is detected in post-mitotic neurons of healthy controls as well, albeit to a lesser extent than in AD patients. This indicates that cell cycle proteins may serve important physiological functions in differentiated neurons. Here, we provide an overview of studies that support a role of cell cycle proteins in DNA repair and neuroplasticity in post-mitotic neurons. Aberrant control of these processes could, in turn, contribute to cell cycle-mediated neurodegeneration. The balance between regenerative and degenerative effects of cell cycle proteins in post-mitotic neurons might change throughout the different stages of AD. In the early stages of AD pathology, cell cycle protein expression may primarily occur to aid in the repair of sublethal double-strand breaks in DNA. With the accumulation of pathology, cell cycle-mediated neuroplasticity and neurodegeneration may become more predominant. Understanding the physiological and pathophysiological role of cell cycle proteins in AD could give us more insight into the neurodegenerative process in AD.

From laryngeal epithelial precursor lesions to squamous carcinoma of the larynx: the role of cell cycle proteins and β-catenin

Novel markers to accurately predict the risk of malignant transformation in laryngeal epithelial precursor lesions (EPL) are needed. We tried to identify some molecular alterations occurring in laryngeal tumorigenesis. In this study, 60 paraffin-embedded EPL and 17 metachronous invasive carcinomas were immunostained for markers associated with proliferation (Ki67), cell cycle control (p53, p21, p16, p27, cyclin D1), and cell adhesion and invasion (laminin and β-catenin). Aberrant expression of p16 and p53 and positivity at cytoplasm for β-catenin and cyclin D1 were detected significantly in EPL with progression to invasive laryngeal carcinoma. All cases with basal and suprabasal reactivity of p53 showed β-catenin overexpression. We found that β-catenin protein expression increased significantly with the grade of dysplasia. This is one of the studies with the largest number of laryngeal EPL and invasive carcinoma studied sequentially. Our data confirm the role of some cell cycle regulatory proteins in the development of laryngeal carcinoma. Cytoplasmic retention of β-catenin in EPL seems to be related with more aggressive biological behavior. Combined increased p53 and cytoplasmic β-catenin protein expression could be biologically important in laryngeal tumorigenesis. Further research is required to clarify the involvement of β-catenin in the mechanism associated with malignant transformation in laryngeal tissues.

Abstract A20: Investigating the role of cell cycle proteins and CDK4/6 inhibitor PD0332991 in the redifferentiation of irreversible dysplasia

Abstract Background: An important strategy in cancer prevention treatment programs is to target the reversal of premalignant disease through re-differentiation. Furthermore, understanding early changes occurring during preneoplastic dysplasia may provide diagnostic and therapeutic insight for cancer prevention programs. Aberrant regulation of the CDK4/6-CyclinD-Rb signaling pathway is one of the most common aberrations found in human cancer; however, there are few published reports describing the status of these cell cycle proteins in preneoplasia. Identifying biomarkers of reversal of dysplasia as well as mechanisms of malignant transformation from preneoplastic disease to cancer will be essential in drug development for chemoprevention strategies. Preclinical mouse models of reversible and refractory dysplasia help elucidate mechanisms responsible for irreversible disease progression and highlight potential targets for its reversal and prevention. This study examined and compared the potential roles of altered expression and/or activation of cell cycle regulatory proteins. Additionally, we examined the effect of a CDK4/6 inhibitor PD0332991 in a system of early dysplasia. Specific Aims: 1. Evaluate expression patterns of cell cycle regulatory network proteins in reversible and irreversible stages of epithelial dysplasia. 2. Test if inhibition of CDK4/6 through PD0332991 can initiate re-differentiation of refractory epithelial dysplasia. 3. Evaluate cell cycle protein expression changes after inhibition of CDK4/6. Methods: A conditional transgenic mouse model, MMTV-tTA/tet-op-TAg (tTA/TAg) demonstrating time dependent reversible and refractory irreversible stages of salivary epithelial dysplasia following down regulation of the initiating oncogene was used. Previous studies demonstrated refractory dysplasia is a field effect without chromosomal rearrangements. Cell cycle regulatory protein expression patterns were compared by western blot and IHC analyses at the 4 (reversible) and 7 (irreversible) month stages. To test the chemopreventive role of CDK4/6 inhibition, PD0332991 inhibitor was administered to at the 7-month (irreversible) stage of dysplasia. Doxycycline was administered with the maximum recommended dose of PD0339221 (150 mg/kg) for 10 days via gavage. Results and Conclusions: Cell cycle proteins were differentially expressed at the reversible 4-month vs. irreversible 7-month time-point. The pattern of expression suggests that irreversible refractory dysplasia is maintained independently from the initiating oncogene due to persistent up-regulation of Cyclin D1 and CDK4/6 with consequent Rb phosphorylation and increased levels of DP-1 and E2F in a self-renewing cyclical manner. Treatment with PD0332991 at the 7 month (irreversible) stage resulted in a significant histological reversal in preliminary studies. The data indicate that cell cycle proteins play an important role in maintenance of dysplasia and are valid targets for cancer chemoprevention. Citation Information: Cancer Prev Res 2011;4(10 Suppl):A20.

CKS Proteins Protect Mitochondrial Genome Integrity by Interacting with Mitochondrial Single-stranded DNA-binding Protein

Cyclin-dependent kinase subunit (CKS) proteins interact with cyclin-dependent kinases (CDKs) with high affinity. Mammalian CKS1 and CKS2 bind CDK1 and CDK2 and partake in the control of cell cycle progression. We identified CKS-interacting proteins by affinity purification followed by mass spectrometry in the human lymphocytic cell line Ramos. Apart from known interactors, such as CDKs, we identified a novel CDK-dependent interaction between CKS proteins and the mitochondrial single-stranded DNA-binding protein (mtSSB). mtSSB bound both CKS1 and CKS2 and underwent CDK-dependent phosphorylation. mtSSB is known to participate in replication of mitochondrial DNA. We demonstrated that mitochondrial morphology and DNA integrity were compromised in cells depleted of both CKS proteins or that had inhibited CDK activity. These features are consistent with the hypothesis of CKS-dependent regulation of mtSSB function and support a direct role of cell cycle proteins in controlling mitochondrial DNA replication.

Differential Expressions of Cyclin D1 Associated with Better Prognosis of Cancers of Ampulla of Vater

Periampullary cancers, the incidence of which increases gradually with industrialization, still pose a significant challenge to clinicians and researchers. Specifically, the role of cell-cycle proteins and tumor suppressor genes in these cancers is not yet clear. Recent studies have revealed that genes and proteins related to cell cycle and apoptosis regulation may be involved in pancreatic carcinogenesis. Tissue samples were obtained from patients with periampullary cancers who underwent surgery at the National Taiwan University Hospital without receiving previous chemotherapy or radiation therapy. All periampullary cancer tissue samples were examined by a pathologist, who was unaware of the parameters to be investigated. A total of 68 patients with periampullary cancers (29 ampulla of Vater cancers (AVCs) and 39 pancreatic ductal cancers (PDCs), including various stages and histological subtypes, were enrolled. The relevant demographic and clinicopathological information was obtained from medical records. Cell-cycle proteins, including p16, Rb, cyclin D1, p53, and E2F1, were analyzed by immunohistochemical staining. Here, significant differences were noted between AVCs and PDCs with regard to the expression of cyclin D1. This corresponded to a poor prognosis in PDCs (P < 0.05); AVCs, on the other hand, showed a relatively high survival rate. There is no obvious statistical difference between the 2 groups with regard to the expression of p16, Rb, p53, and E2F1. The study also revealed that cyclin D1 plays different roles in the carcinogenesis of AVCs and PDCs. The expression of cyclin D1 is more often correlated with prognosis in AVCs than in PDCs, and may serve as a biomarker for the disease.

Role of Cell Cycle Proteins in CNS Injury

Following trauma or ischemia to the central nervous system (CNS), there is a marked increase in the expression of cell cycle-related proteins. This up-regulation is associated with apoptosis of post-mitotic cells, including neurons and oligodendrocytes, both in vitro and in vivo. Cell cycle activation also induces proliferation of astrocytes and microglia, contributing to the glial scar and microglial activation with release of inflammatory factors. Treatment with cell cycle inhibitors in CNS injury models inhibits glial scar formation and neuronal cell death, resulting in substantially decreased lesion volumes and improved behavioral recovery. Here we critically review the role of cell cycle pathways in the pathophysiology of experimental stroke, traumatic brain injury and spinal cord injury, and discuss the potential of cell cycle inhibitors as neuroprotective agents.

DNA damage induces cdk2 protein levels and histone H2B phosphorylation in SH-SY5Y neuroblastoma cells

DNA damage and activation of the cell cycle have been implicated in numerous neurodegenerative diseases, including Alzheimer disease, Parkinson's disease, and amyotrophic lateral sclerosis. To better understand the role of cell cycle proteins in DNA-damage induced neuronal cell death, we examined various cell cycle proteins during camptothecin-induced death of human neuroblastoma cells. We report a rapid induction of p53 and increased expression of p21, concurrent with reduced levels of many cell cycle proteins that regulate G1 to S phase cell cycle progression. However, we found increased levels of cdk2 and cyclin E, and formation of a cyclin E-cdk2-p21 protein complex. DNA damage failed to induce activation and progression of the cell cycle. Finally, camptothecin-induced neuronal cell death occurred concurrent with phosphorylation of histone H2B. Pretreatment of cells with cdk inhibitor olomoucine impeded cdk2-cyclin E accumulation, but not the induction of p53. Olomucine concurrently delayed histone H2B phosphorylation, caspase-3 activation and cell death. These findings suggest that DNA-damage of differentiated neuroblastoma cells induces a rapid p53-mediated inhibition of cell cycle progression and induction of cdk2-cyclin E, followed by caspase-3 activation, phosphorylation of histone and cell death.

Role of cell‐cycle regulators in lung cancer

Lung cancer is the leading cause of cancer death worldwide. Histologically, 80% of lung cancers are classified as non-small-cell lung cancer (NSCLC), and the remaining 20% as small-cell lung cancer (SCLC). Lung carcinoma is the result of molecular changes in the cell, resulting in the deregulation of pathways controlling normal cellular growth, differentiation, and apoptosis. This review summarizes some of the most recent findings about the role of cell-cycle proteins in lung cancer pathogenesis and progression.

Cell Cycle Regulators Modulating Con A Mitogenesis and Apoptosis in Low-Dose Radiation-Exposed Mice

Low doses of ionizing radiation (LDR) are reported to induce transient changes in mouse lymphocytes, such as enhanced response to polyclonal T cell mitogens, increased expression of heat shock proteins, and p53. We evaluated the role of cell cycle proteins and apoptosis in lymphocytes of C57BL/6 mice exposed to 20 cGy fractionated LDR. We found an enhanced cell proliferation in response to the mitogen concanavalin A (con A). The expression of several cell cycle and apoptosis-related intracellular and extracellular proteins was analyzed by flowcytometry following labeling with specific antibodies. An increased response to con A was accompanied by an increase in the expression of proliferating cell nuclear antigen (PCNA) and cyclins D and A. The expression of cyclin B did not change significantly. In 20 cGy-exposed C57BL/6 mice, the caspase activity and apoptosis were reduced in con A-stimulated spleen cells as compared to sham controls. The expression of Fas and Fas ligand was analyzed by labeling with specific antibodies followed by flowcytometry. There was no change in the expression of Fas and Fas ligand. The change in the mitochondrial transmembrane potential was followed by labeling the cells with the dye 5,5',6,6',-tetrachloro-1,1,3,3-tetraethylbenzimidazolcarbocyanine iodide (JC-I) and analyzing by flowcytometry. Mitochondrial stability was increased in spleen cells of LDR-treated mice. These data suggest that LDR induces augmentation of mitogenic response by modulation of expression of cyclins and the mitochondrial membrane potential leading to reduced apoptosis.

Characterization of DNA demethylation in normal and cancerous cell lines and the regulatory role of cell cycle proteins in human DNA demethylase activity.

DNA methylation/demethylation constitutes a major consequence in all biological processes involving transcription, differentiation, development, DNA repair, recombination, and chromosome organization. Our earlier studies established that demethylation of CpG rich sequence by human DNA demethylase activity (5-methylcytosine-DNA glycosylase (5MeC-DNA glycosylase)) resembles "base excision DNA repair activity" and creates single-strand breaks on DNA that is associated with proliferating cell nuclear antigen (PCNA). Here in this report, we have identified differential DNA demethylation targets (hemi-methylated vs. fully-methylated) in normal cell lines and cancerous cell lines, and a shortened G(0)/G(1) resting time in cancerous cell lines than the normal cell lines. We have identified that in normal HFL1 fibroblast cell line, DNA demethylase activity targets hemi-methylated CpG specific sites on DNA. This normal cell line DNA demethylase activity associates with PCNA immune complex that is inhibited by CDKI proteins p21(waf1)/Gadd45alpha and Gadd45beta. While in cancerous LnCap and BT20 cell lines DNA demethylase activity targets fully-methylated CpG specific sites on DNA. This cancer cell line DNA demethylase activity is not associated with PCNA immune complex and is not inhibited by CDKI proteins p21(waf1)/Gadd45alpha and Gadd45beta. We have also identified that the fully-methylated CpG specific DNA demethylase activity from cancerous cell lines to associate with p300/CBP protein. These significant observations of variable targets of DNA demethylation and alternate partner proteins for DNA demethylase activity in cancerous cell lines are discussed in terms of double-strand DNA breaks versus single-strand DNA breaks and their role in the exit of G(1)/G(2) cell cycle stages. Also, the inability of cell cycle regulatory proteins like PCNA, p21(waf1), and Gadd45 to control DNA demethylase activity in cancerous cell lines is discussed in terms of accelerated G(1)/G(2) cell cycle stage exit to facilitate unregulated cellular proliferation, loss of control of chromosomal organization, and the development of oncogenesis in cancerous cell lines.

The role of cell cycle proteins in Glomerular disease

Although initially identified and characterized as regulators of the cell cycle and hence proliferation, an extended role for cell cycle proteins has been appreciated more recently in a number of physiologic and pathologic processes, including development, differentiation, hypertrophy, and apoptosis. Their precise contribution to the cellular response to injury appears to be dependent on both the cell type and the nature of the initiating injury. The glomerulus offers a remarkable situation in which to study the cell cycle proteins, as each of the 3 major resident cell types (the mesangial cell, podocyte, and glomerular endothelial cell) has a specific pattern of cell cycle protein expression when quiescent and responds uniquely after injury. Defining their roles may lead to potential therapeutic strategies in glomerular disease.

Cell cycle protein expression in proliferating microglia and astrocytes following transient global cerebral ischemia in the rat

Cerebral ischemia induces microglial and astroglial activation, which may play a crucial role in the development of ischemic neuronal damage. In this study, we examined the role of cell cycle proteins in glial proliferation in the hippocampus following 10 min of global cerebral ischemia in the rat. Proliferating cells were identified with immunostaining for proliferating cell nuclear antigen (PCNA), and glial cells were visualized with immunostaining for microglial response factor-1 (microglia/macrophages) and glial fibrillary acidic protein (astrocytes). Expression of cyclin D1 and cyclin-dependent kinase-4 was also examined with double label immunohistochemistry. Proliferating cells in the CA1 region after ischemia consisted of microglia and much fewer astrocytes. Microglial activation and proliferation (7.6-fold increase in number after 7 days) were preceded by an increase in PCNA-positive microglia; 83% of microglia were PCNA-positive after 2 days. Astrocytes increased by 1.8-fold after 7 days, and only 6% of astrocytes became PCNA-positive by day 7. Cyclin D1 and cyclin-dependent kinase-4 immunoreactivity appeared in these glial cells in parallel with the expression of PCNA. The findings suggest that the accumulation of brain macrophages elicited by transient cerebral ischemia is caused predominantly by activation and proliferation of resident microglia through the upregulation of cell cycle proteins.

Podocytes, parietal cells, and glomerular pathology: the role of cell cycle proteins.

Podocytes are highly differentiated cells that play a central role in glomerular function. Cell cycle quiescence sustained by the tight regulation of cell cycle molecules seems to be the basis of podocyte differentiation, as reflected in reciprocal expression of cyclins and cyclin kinase inhibitors (CKIs) during glomerulogenesis, concurrent with the expression of podocyte-specific markers. In addition, cell cycle re-entry by podocytes, accompanied by down-regulation of CKIs, leads to either nuclear division without cytokinesis or to marked cell proliferation, both of which result in heavy proteinuria and progressive glomerulosclerosis. These observations suggest that proper cell cycle regulation in podocytes is pivotal for their role in glomerular function and that dysregulation of this system plays a role in glomerular pathology. This review discusses the role of cell cycle molecules in the differentiation, function, and pathology of podocytes.

Altered subcellular distribution of transcriptional regulators in response to Aβ peptide and during Alzheimer's disease

Recent studies have shown that various cell cycle proteins are expressed in post-mitotic neurons within affected brain regions during Alzheimer's disease (AD). Cell cycle proteins have been proposed to function in mechanisms of neuronal cell death during AD. To further explore the role of cell cycle proteins in neurodegeneration associated with AD, we utilized PC12 cells to examine the subcellular distribution of cell cycle transcriptional regulators, including the retinoblastoma gene product (pRb), E2F1 and FAC1, during β-amyloid (Aβ)-induced neurodegeneration. Moreover, we examined the immunolocalization of pRb and E2F1 in non-demented control and AD brain tissue. We found that pRb exhibited increased levels of Ser795 phosphorylation in response to Aβ in the nucleus of PC12 cells and also in the nucleus of a subset of neurons during AD. E2F1 was distributed throughout the cytoplasm and neurites of PC12 cells in response to Aβ and in the cytoplasm of cells in AD brain. FAC1 exhibited a rapid redistribution from the cytoplasm to the perinuclear region in PC12 cells treated with Aβ. These data indicate that altered phosphorylation and subcellular distribution of transcriptional regulators occur in response to Aβ-induced neurotoxicity and during AD.

Cell cycle control in growth plate chondrocytes.

Growth of endochondral bones is regulated by the coordinated proliferation and differentiation of chondrocytes in the epiphyseal growth plates. Many skeletal diseases are caused by pathogenic disruptions of these two processes. While the intracellular mechanisms regulating chondrocyte proliferation and differentiation are poorly understood, recent evidence from studies using genetically altered mice and from experiments in cultured chondrocytes point to a prominent role of cell cycle proteins in this context. This article summarizes our current understanding of the expression, regulation, and function of cell cycle genes in chondrocytes.